Exposure to growth hormone is associated with hepatic up-regulation of cPLA2α and COX
Introduction
Growth hormone (GH) has a prominent role in the stimulation of body growth and metabolism. It also influences cardiovascular, renal, reproductive and immune functions and regulates body composition (Chhabra et al., 2011; Chia, 2014; Guevara-Aguirre et al., 2018). Accordingly, GH therapy restores growth velocity in GH-deficient children and improves body composition, exercise capacity, skeletal integrity and quality of life in GH-deficient adults (Chia, 2014; Pekic et al., 2017). It exerts its actions on different tissues through its membrane receptor, which triggers multiple intracellular signaling pathways and induces the expression of different genes. Insulin-like growth factor-1 (IGF-1) is a key transcriptional target of GH in liver and other tissues and is the principal mediator of many GH actions (Chhabra et al., 2011; Chia, 2014; Guevara-Aguirre et al., 2018).
Elevated levels of GH and IGF-1 promote hyperplasia, hypertrophy and alterations in metabolism that may lead to different pathologies. In particular, the potential role of GH and IGF-1 in the genesis and progression of cancer was recognized several years ago and has been the focus of interest in recent years (Jenkins et al., 2006; Perry et al., 2013; Pekic et al., 2017; Guevara-Aguirre et al., 2018, Boguszewski and Boguszewski, 2019). As these peptides promote cell proliferation, cell movement and angiogenesis and have anti-apoptotic effects, dysregulation of GH/IGF-1 axis may be linked to cancer occurrence or promotion (Chhabra et al., 2011; Pekic et al., 2017). Indeed, evidence from epidemiologic, animal and in vitro studies support the notion that the state of the GH/IGF-1 axis influences carcinogenesis (Chhabra et al., 2011; Pekic et al., 2017, Boguszewski and Boguszewski, 2019). In patients with hepatocellular carcinoma (HCC), increased GH expression was observed in tumors in comparison to non-tumoral surrounding tissue and GH levels were significantly associated with tumor size, higher histological grade and poor patient survival (Kong et al., 2016). Moreover, these authors have shown in vivo that autocrine expression of GH promoted oncogenicity and HCC xenograft growth (Kong et al., 2016).
Liver is a major target for GH action, where GH signaling is necessary for normal hepatocyte proliferation and liver regeneration (Chia, 2014). In rodents, a role for GH in liver tumorigenesis is particularly evident. GH deficiency was reported to suppress carcinogen-induced liver tumor development in mice (Bugni et al., 2001). In contrast, transgenic mice overexpressing GH are more susceptible to hepatocarcinogenesis. These mice exhibit hepatomegaly associated with hypertrophy and hyperplasia of hepatocytes since young ages (Snibson et al., 1999; Miquet et al., 2013; Martinez et al., 2016). A state of sustained increase in hepatocyte turnover and chronic inflammation precedes the development of liver tumors, including HCC, which are most commonly observed within a year (Quaife et al., 1989; Orian et al., 1990; Snibson et al., 1999; Bartke, 2003; Kopchick et al., 2014). The hepatocarcinogenesis observed in GH-transgenic mice seems to be a direct consequence of elevated GH levels, since IGF-1-transgenic mice do not develop liver tumors (Quaife et al., 1989; Bartke, 2003; Kopchick et al., 2014). Moreover, the liver pathology would not be due to the expression of the transgene in the liver or to the abnormal actions of heterologous GH, since transgenic mice for GH releasing-hormone present similar liver abnormalities (Bartke, 2003). Importantly, we have reported the dysregulation of several oncogenic pathways in young adult GH-transgenic mice (Miquet et al., 2008, 2013; Bacigalupo et al., 2019), some of which are altered since early ages (Martinez et al., 2016). The molecular alterations and the preneoplastic liver pathology observed in these animals are similar to those present in patients at high risk of developing hepatic cancer (Snibson et al., 1999; Snibson, 2002; Miquet et al., 2013).
The process of hepatic carcinogenesis involves sequential events including chronic inflammation, hyperplasia of hepatocytes, dysplasia and, finally, malignant transformation (Snibson et al., 1999; Wu, 2006; Schlageter et al., 2014; Llovet et al., 2016). Several lines of evidence point to an important role of mediators of inflammation, such as prostaglandins (PGs), in liver carcinogenesis (Wu, 2006; Wang and Dubois, 2010; Zang et al., 2017; Kim et al., 2018).
In a study using transgenic mice overexpressing ovine GH under the control of the metallothionein promoter (Mt-oGH), the long-term exposure to GH was associated with elevated levels of arachidonic acid (AA) in the liver and of one of its metabolites, prostaglandin E2 (PGE2), in serum (Oberbauer et al., 2011). AA is the long chain polyunsaturated fatty acid most abundant in biological membranes. Its release from membrane-bound phospholipids for subsequent prostaglandin synthesis is primarily catalyzed by the cytosolic phospholipase A2α (cPLA2α). AA is then transformed to eicosanoids by the action of cyclooxygenases COX1 and COX2 (Niknami et al., 2009; Dennis et al., 2011; Kim et al., 2018). Higher hepatic mRNA levels of COX enzymes have also been described in Mt-oGH mice (Oberbauer et al., 2011).
Altered metabolism of AA and lipid mediators have been associated with pathogenesis of cancers, including HCC (Subbaramaiah and Dannenberg, 2003, Chi-Man Tang et al., 2005, Cervello and Montalto, 2006, Nakanishi and Rosenberg, 2006, Wu, 2006, Breinig et al., 2007, Martín-Sanz et al., 2010, Wang and Dubois, 2010, Kim et al., 2018). Therefore, the enhanced AA metabolism reported in Mt-oGH transgenic mice may constitute an unrecognized molecular mechanism implicated in the liver pathology that develops in mice overexpressing GH. However, in that report (Oberbauer et al., 2011) no histopathological study of the liver was performed and the possible association of these findings with the hepatocarcinogenesis process was not explored.
In order to investigate if alterations in AA metabolism may be associated with the process of liver carcinogenesis observed in GH-transgenic mice, we used transgenic mice overexpressing bovine GH, in which we have already described that several signaling mediators involved in cell proliferation are exacerbated in young animals (Miquet et al., 2008, 2013; Martinez et al., 2016). In the present work, we have determined the expression of key enzymes involved in AA metabolism and production of PGs, namely, cPLA2α, COX1 and COX2, in the liver of young adult GH-transgenic mice, which exhibit preneoplastic liver pathology, and in old animals, which display marked hepatic alterations and frequently develop tumors. In addition, we assessed if a short-term continuous administration of GH could induce changes in cyclooxygenases liver expression.
The results of the present study contribute to better characterize the molecular alterations associated with sustained exposure to high GH levels that may play a prominent role in the process of liver carcinogenesis. The elucidation of the molecular mechanisms underlying GH action may have important implications in understanding human health and disease (Chia, 2014).
Section snippets
Transgenic mice overexpressing growth hormone
Transgenic PEPCK-bGH mice used express the bovine GH gene linked to control sequences of the rat phosphoenolpyruvate carboxykinase (Pepck). These mice exhibit accelerated postweaning growth that lead to a significant increase in body weight and organomegaly. Normal-sized siblings were used as controls. Breeding system, animal housing and feeding conditions have been previously described (Mcgrane et al., 1988; Miquet et al., 2013).
Young (2 months old) and old (10–13 months old) animals were
Liver macroscopic analysis
Exposure to high GH levels in mice promotes hypertrophy and hyperplasia of hepatocytes that lead to hepatomegaly and, frequently, to liver tumor development (Orian et al., 1990; Snibson et al., 1999; Snibson, 2002). The disproportional growth of liver is evidenced even in absence of preneoplastic liver lesions (Martinez et al., 2016). In accordance with previous reports, young adult GH-transgenic mice used in this work exhibited hepatomegaly, manifested by a higher liver to body weight ratio
Discussion
GH is currently administered to children with retarded growth, related or not to GH deficiency. In adults, it is also prescribed due to its beneficial effects over metabolism and neuronal and reproductive function. GH abuse has also been reported since it modifies body composition, increasing muscle mass and decreasing fat depots (Kemp and Frindik, 2011; Laron, 2011). In humans, there are no conclusive studies that directly link GH treatment with tumor development. In fact, GH replacement
Funding
This work was supported by the Universidad de Buenos Aires (grant numbers 20020120200122BAto JGM, 20020170100551BAto AIS); the Agencia Nacional de Promoción Científica y Tecnológica (grant number PICT-2015-1100 to JGM) and the National Institute on Aging of the National Institutes of Health (grant number R01AG019899to AB).
CRediT authorship contribution statement
Verónica G. Piazza: Investigation, Writing - original draft, Visualization. María E. Matzkin: Investigation, Writing - review & editing. Nadia S. Cicconi: Investigation. Nadia V. Muia: Investigation. Sofía Valquinta: Investigation. Gregorio J. Mccallum: Investigation. Giannina P. Micucci: Investigation. Thomas Freund: Investigation. Elsa Zotta: Investigation. Lorena González: Resources, Writing - review & editing. Mónica B. Frungieri: Resources, Writing - review & editing. Yimin Fang:
Declaration of competing interest
All the authors declare no conflict of interest.
Acknowledgements
We thank Samuel A McFadden (Southern Illinois University School of Medicine) for his laboratory assistance.
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Cited by (0)
- 1
Instituto de Nanobiotecnología (NANOBIOTEC), CONICET-Universidad de Buenos Aires, Buenos Aires, Argentina.
- 2
Hospital Italiano de Buenos Aires, Servicio de Farmacia, Buenos Aires, Argentina.
- 3
Medizinisches Versorgungszentrum Labor28, Berlin, Germany.